Field of the Invention
This invention relates to nucleic acid (NA) detection, and in particular, it relates to a method that employs ultra-specific probe, isotachophoresis (ITP), and enzyme amplification to achieve rapid, highly-sensitive, and highly-specific NA detection.
Description of Related Art
An ultra-specific probe which can discriminate single nucleotide mutations with high-specificity is described in Optimizing the specificity of nucleic acid hybridization, D. Y. Zhang, S. X. Chen, P. Yin, Nature Chemistry (2012), Vol. 4, 208-214, March 2012 (published online Jan. 22, 2012) (“Zhang et al. 2012”). As shown in FIG. 1 (adapted from FIG. 2 of the above article; in FIG. 1, shading patterns schematically represent the sequences where the same shading pattern represent the same sequence, and the dots between two sequences indicate that the two sequences are complementary to each other), the ultra-specific probe used in this technology (called “toehold exchange probe”) is a double strand-DNA (PC) consisting of a protector strand (P) and a complement strand (C). The complement strand C has a longer sequence than the protector strand P. A part of the complement strand C is 100% complementary with the target sequence X. Because the affinity between the target sequence X and the complement strand C is higher than that between the protector strand P and the complement strand C, in the presence of target X, the protector strand P is displaced and a target-C double strand is formed. However, if the DNA to be tested has more than one mutation relative to the target sequence (i.e. it is a non-target, denoted “spurious target S” in FIG. 1), the affinity between the non-target S and the complement strand C is lower than that between the protector strand P and the complement strand C, and the strand displacement (or exchange) reaction does not occur. By detecting the target-C double strand or the isolated protector strand P, the amount of the target sequence X can be measured with high-specificity.
Isotachophoresis (ITP) is an electrophoresis technique that uses two buffers including a high mobility leading electrolyte (LE) and a low-mobility trailing electrolyte (TE). In peak-mode ITP, sample species bracketed by the LE and TE focus into a narrow TE-to-LE interface. Due to the high concentration of sample species in a small volume at the interface, high efficiency (rapid) molecular-molecular interaction can occur.
On-chip isotachophoresis (ITP) is a technology that can realize ultra-rapid reactions by focusing the sample in the solution. Isotachophoresis with ionic spacer and two-stage separation for high sensitivity DNA hybridization assay, Charbel Eid, Giancarlo Garcia-Schwarz and Juan G. Santiago, Analyst (2013), 138, 3117-3120 (“Eid et al. 2013”), describes a technique of ultra-rapid DNA hybridization and subsequent single strand- and double strand-DNA separation using ITP. As illustrated in FIG. 2, adopted from FIG. 1 of Eid et al. 2013, ITP is used to enhance NA hybridization and an ionic spacer molecule is used to subsequently separate the reaction product. In the first stage, the probe and target are focused and mix rapidly in free solution under ITP. The reaction mixture then enters a region of the microfluidic channel containing a sieving matrix, which allows the spacer ion to overtake the slower double strand NA and separate it from the single strand NA.
Eid et al. 2013 describes its FIG. 1 as follows (p. 3118, left column): “Our assay includes a spacer ion with intermediate mobility which forms a plateau region between the LE and TE, thereby creating two sharp interfaces between the LE and spacer and between the spacer and TE. FIG. 1a demonstrates the steps in our reaction-separation assay. First, we leverage ITP to focus the probe and target molecules and accelerate second-order hybridization kinetics (time 1). The second and third stages of the assay, denoted respectively by t2 and t3, employ a linear sieving matrix to separate the reaction products. The channel initially contains two LE regions in series, as shown in FIG. 1b. LE1 includes no sieving matrix, while LE2 includes a sieving matrix. The sieving matrix primarily affects mobility of DNA molecules relative to small ions. In the LE1 region, spacer ions have an electrophoretic mobility lower than that of the probe, target, and probe-target complexes. This enables simultaneous rapid mixing and preconcentration of the probe and its target. Upon entering LE2, the spacer ions overtake the now slower target and probe-target complex. The spacer has sufficient initial concentration to quickly form a plateau ITP region which separates excess probes from probes hybridized to target molecules. In this final stage, the excess probe molecules continue to focus between the LE and the spacer, while the probe-target complexes focus in a separate ITP zone between the spacer and the TE. This enables sensitive detection of the probe-target complexes in the absence of unhybridized fluorescent probe molecules.”